Facultative Apomicts Research Articles

Registration of ‘Mallard’ Kentucky Bluegrass

Kentucky bluegrass is a pseudogomous facultative apomict, requiring viable pollen to stimulate seed set. Since Kentucky bluegrass is a facultative apomict, low levels of sexuality occur that allow for hybridization and genetic recombination. ‘Mallard’ (Reg. No. CV‐100, PI 632598) Kentucky bluegrass (Poa pratensis L.) is a turf‐type cultivar released by the Pennington Seed (Athens, GA) in 2002. Mallard was selected as an open‐pollinated progeny of ‘Unique’ Kentucky bluegrass. Mallard is comparable with ‘Midnight’ (standard) in many turf characteristics, including turf quality and disease resistance. Mallard is significantly better in drought resistance compared with Unique (parent). The combination of these traits makes Mallard a unique, widely adapted, functional cultivar that should perform well in home lawns, golf courses, and municipalities in the Northeast, transition, North Central, and Upper West, and Mountain regions.

Residual sexuality and its seasonal variation in natural apomictic Paspalum notatum accessions

Traditionally, tetraploid Paspalum notatum was considered an obligate or a facultative apomict according to cytoembryological analyses. The degree of facultativeness was usually determined by the relative amount of mature ovules bearing aposporous or meiotic (sexual) embryo sacs, or both together. We established, through progeny tests conducted with the aid of AFLP markers, the degree of residual sexuality expressed in four selected biotypes. The results showed it to be substantially and significantly lower than predicted by previous embryological analyses for the same biotypes. Moreover, the lowest expression of residual sexuality was coincident with maximum flowering period. Seed development in facultative apomictic P. notatum shows a definite bias against meiotic embryo sacs.

Cytogenetic peculiarities of the genesis of apical meristem cells in case of gametophytic apomixis (with reference to autonomous Asteraceae apomicts)

The cytogenetic peculiarities of the genesis of apical meristem cells in apomicts were analyzed using some Asteraceae species as an example. It has been revealed that the frequency of aneu- and mixoploids is so high among the plants of these species (up to 30–60% of all plants studied or their progeny) that there is every reason to say that their occurrence in apomicts is regular rather than spontaneous. It has been demonstrated that microgametophyte in aposporous facultative apomict Pilosella officinarum is a relatively stable element of the seed reproduction system in terms of the ploidy level.

Apomixis in hawkweed: Mendel's experimental nemesis

Mendel used hawkweeds and other plants to verify the laws of inheritance he discovered using Pisum. Trait segregation was not evident in hawkweeds because many form seeds asexually by apomixis. Meiosis does not occur during female gametophyte formation and the mitotically formed embryo sacs do not require fertilization for seed development. The resulting progeny retain a maternal genotype. Hawkweeds in Hieracium subgenus Pilosella form mitotic embryo sacs by apospory. The initiation of sexual reproduction is required to stimulate apospory in ovules and to promote the function of the dominant locus, LOSS OF APOMEIOSIS, which stimulates the differentiation of somatic aposporous initial (AI) cells near sexually programmed cells. As AI cells undergo nuclear mitosis the sexual pathway terminates. The function of the dominant locus LOSS OF PARTHENOGENESIS in aposporous embryo sacs enables fertilization-independent embryo and endosperm development. Deletion of either locus results in partial reversion to sexual reproduction, and loss of function in both loci results in reversion to sexual development. In these apomicts, sexual reproduction is therefore the default reproductive mode upon which apomixis is superimposed. These loci are unlikely to encode factors critical for sexual reproduction but might recruit the sexual pathway to enable apomixis. Incomplete functional penetrance of these dominant loci is likely to lead to the generation of rare sexual progeny also derived from these facultative apomicts.

Apospory appears to accelerate onset of meiosis and sexual embryo sac formation in sorghum ovules.

BackgroundGenetically unreduced (2n) embryo sacs (ES) form in ovules of gametophytic apomicts, the 2n eggs of which develop into embryos parthenogenetically. In many apomicts, 2n ES form precociously during ovule development. Whether meiosis and sexual ES formation also occur precociously in facultative apomicts (capable of apomictic and sexual reproduction) has not been studied. We determined onset timing of meiosis and sexual ES formation for 569 Sorghum bicolor genotypes, many of which produced 2n ES facultatively.ResultsGenotype differences for onset timing of meiosis and sexual ES formation, relative to ovule development, were highly significant. A major source of variation in timing of sexual germline development was presence or absence of apomictic ES, which formed from nucellar cells (apospory) in some genotypes. Genotypes that produced these aposporous ES underwent meiosis and sexual ES formation precociously. Aposporous ES formation was most prevalent in subsp. verticilliflorum and in breeding lines of subsp. bicolor. It was uncommon in land races.ConclusionsThe present study adds meiosis and sexual ES formation to floral induction, apomictic ES formation, and parthenogenesis as processes observed to occur precociously in apomictic plants. The temporally diverse nature of these events suggests that an epigenetic memory of the plants' apomixis status exists throughout its life cycle, which triggers, during multiple life cycle phases, temporally distinct processes that accelerate reproduction.

Analysis of breeding systems, ploidy, and the role of hexaploids in three Hypericum perforatum L. populations

Hexaploid seeds are produced by predominantly tetraploid populations of Hypericum perforatum, but the fate of hexaploid seedlings and their reproductive behavior have not been closely examined. We used flow cytometry to analyze single seeds and individual plant samples of three accessions of H. perforatum to determine ploidy levels and reproductive pathways. Seed samples of all three accessions were facultative apomicts, with tetraploid cytotype predominant (85–91%) and a lower frequency of hexaploids (9–14%), with diploids (5%) detected in only one population. Seedling populations consisted of tetraploids (87–97%) and hexaploids (3–13%). Hexaploid embryos are most likely generated by a 2 n gamete of the tetraploid and fertilized by a normal, reduced tetraploid male gamete. These hexaploids are expected to produce unbalanced gametes because they possess chromosome complements that include two triploid sets originally derived from two different species. The observation that some tetraploid seeds had endosperm with high cellular DNA content indicates that some unbalanced male gametes produced by hexaploids were evidently viable and could effect fertilization. Whether this mechanism is also true for egg cells or whether the hexaploids are capable of producing unreduced embryo sacs is uncertain. Because of severe reproductive difficulties, hexaploid seedlings may play a very minor role in gene flow and the further evolution of H. perforatum. The likelihood that hexaploids will evolve to types with an increased frequency of bivalent paring in meiosis is relatively low. However, hexaploids may include novel chemotypes, which could be vegetatively propagated if valuable, medicinal types can be identified among them.

Apomixis in the Sugar Beet Reproduction System

Research on five lines of sugar beet with a tendency towards apomixis showed the presence of facultative apomicts among the studied plants (2.4%). Facultative agamospermy was detected by isozyme analysis and by nuclear DNA amount estimation using flow cytometry. Genetic segregation according to isozymes in seed progenies showed the presence of meiotic agamospermy; its probable mechanism was normal meiosis in tetraploid cells of the female archespore. The occurrence of cytologically unreduced male gametophytes was confirmed in 21% of the plants by the indirect method of determining ploidy level from the number of pore regions in mature pollen grains. These studies identified homozygotic sublines with a tendency towards apomixis, proposed for practical breeding at the Kutno Sugar Beet Breeding Company Ltd., to be included in breeding experiments as components of heterotic hybrids.

Apomixis and sexuality in the Potentilla argentea complex III. Euploid and aneuploid derivatives (including trisomics) of some apomictic biotypes

Three apomictic biotypes belonging to the Potentilla argentea complex formed the basis of this investigation, one of them diploid, one tetraploid and one hexaploid. The effects of chromosome doubling and other changes in chromosome number were studied. Hybrids between the same biotypes were discussed in a previous paper. The diploid was a facultative apomict, but autotetraploids produced by colchicine treatment were totally sexual. This was also true of autotriploids, probably also of trisomics and other aneuploids. Five out of seven possible primary trisomics were obtained; they were morphologically well separated from each other and from the diploid. As shown by the result of test-crosses, facultative apomixis was restored in “recombined diploids” in progenies from triploids and trisomics. This indicates that apomixis is partly dependent on homozygosity for recessive genes, but that also the degree of ploidy as well as chromosomal and genic balance directly influence the mode of reproduction. — In the more strictly apomictic tetraploid (2n= 28) biotype, unreduced egg cells were fertilized in exceptional cases, whereas the chromosome-doubled plants formed some aberrants by fertilization of reduced egg cells. However, an “autotriploid” (2n= 42) formed half of its offspring by fertilization of unreduced and reduced egg cells. Aberrants with 2n= 35 and 2n= 63 were obligate apomicts. — The hexaploid was obligately apomictic, whereas the poorly fertile plants resulting from chromosome doubling gave rise to one plant with further increased chromosome number; thus, even here apomixis was interfered with to some extent. — The concluding discussion also deals with problems of mitotic and meiotic instability, crossing barriers in pseudogamous taxa, and viewpoints on the mechanisms and regulation of apomixis.

Apomixis and sexuality in the Potentilla argentea complex II. Crosses within the complex

The Potentilla argentea complex is mainly built up by apomicts on various levels of ploidy. In a previous paper, the results of crosses with other species were dealt with. Here, an account is given of crosses between taxa within the argentea complex. Most crosses involved a diploid, a tetraploid and a hexaploid biotype. The first-mentioned is a facultative apomict, whereas its autotetraploid is wholly sexual. The tetraploid biotype forms aberrants at a low frequency, whereas the hexaploid seems to be entirely constant. Natural tetraploids may have originated from crosses between the more widely distributed diploid (“P. argentea”) and hexaploid (“P. neglecta”) biotypes. The F1 hybrids were in many cases vigorous but had always a reduced seed set as compared with the parents. Crosses between sexual autotetraploid and apomictic tetraploid resulted in sexual F1 plants, whereas F1 hybrids from crosses between sexual autotetraploid and hexaploid apomicts showed a varying mode of reproduction. From crosses between apomictic tetraploid and hexaploid biotypes, one pentaploid and some heptaploid hybrids were obtained. The heptaploids, resulting from fertilization of unreduced egg cells, were apomictic, although two plants with further increased chromosome numbers were found in in the offspring. The pentaploid hybrid reproduced only by fertilization of unreduced egg cells. This mode of reproduction prevailed even in the next generation, leading to the formation of weak high-polyploid plants.

A Continent‐Wide Clone: Population Genetic Variation of the Invasive PlantHieracium aurantiacum(Orange Hawkweed; Asteraceae) in North America

We investigated the population genetic structure of the invasive plant Hieracium aurantiacum (Asteraceae), a facultative apomict. We generated amplified fragment length polymorphism fingerprints for H. aurantiacum samples from across its invasive range in North America (N ¼ 226) and from six other North American native and invasive Hieracium species (N ¼ 60). Almost no genetic variability was found in the North American H. aurantiacum across locations from Alaska and Oregon to Pennsylvania and Ontario (clonal diversity ¼ 0:035). In contrast, other Hieracium species showed a range of clonal diversities (range ¼ 0:154‐1:0). The single H. aurantiacum genotype that dominated the North American invaded range was identical to a sample from the native range (Czech Republic), where low genetic diversity has also been reported. However, we did find evidence of hybridization between H. aurantiacum and at least one other nonnative Hieracium species in North America, indicating that the generation of novel hybrid genetic combinations may be an important factor in this invasive group of Hieracium taxa. Our findings suggest that sexual recombination and genetic diversity are not essential for successful plant invasion and that phenotypic plasticity alone may provide the flexibility necessary for the establishment of H. aurantiacum in diverse habitats.

Enriching Ploidy Level Diversity: the Role of Apomictic and Sexual Biotypes of Hieracium subgen. Pilosella (Asteraceae) that Coexist in Polyploid Populations

The capacity to generate variation in ploidy and reproductive mode was compared in facultatively apomictic versus sexual maternal plants that coexist in two model populations. The population structure was studied in polyploid hybrid swarms comprised of Hieracium pilosella (usually sexual, less commonly apomictic), H. bauhini (apomictic), and their hybrids (sexual, apomictic, or sterile). Relationships among established biotypes were proposed on the basis of their DNA ploidy level/chromosome number, reproductive mode and morphology. Isozyme phenotypes and chloroplast DNA haplotypes were assayed in the population that was richer in hybrids. The reproductive origin of seed progeny was identified in both sexual and apomictic mothers, using alternative methods: the karyological, morphological and reproductive characters of the cultivated progeny were compared with those of respective mothers, or flow cytometric seed screening was used. In both populations, the progeny of sexual mothers mainly retained a rather narrow range of ploidy level/chromosome number, while the progeny of facultatively apomictic mothers was more variable. The high-polyploid hybrids, which had arisen from the fertilization of unreduced egg cells of apomicts, mainly produced aberrant non-maternal progeny (either sexually and/or via haploid parthenogenesis). Apparently, such versatile reproduction resulted in genomic instability of the recently formed high-polyploid hybrids. While the progeny produced by both true apomictic and sexual mothers mostly maintained the maternal reproductive mode, the progeny of those ‘versatile’ mothers was mainly sexual. Herein, we argue that polyploid facultative apomicts can considerably increase population diversity.

Ploidy level selection during germination and early stages of seedling growth in the progeny of allohexaploid facultative apomict,Hieracium rubrum (Asteraceae)

Selection within progeny of a facultative apomict,Hieracium rubrum, was studied using flow cytometry of embryos in seeds (a modified method of Flow Cytometric Seed Screen) and seedlings. Flow cytometric screening of particular progeny classes was based on distinct ploidy categories, reflecting the way of their origin.

A modified method of flow cytometric seed screen simplifies the quantification of progeny classes with different ploidy levels

Flow cytometric analysis of ten bulked seeds is proposed to quantify particular embryo ploidy classes in Hieracium. The method is recommended 1) for the detection and quantification of residual sexuality in facultative apomicts, which can generate progeny from heteroploid crosses, 2) for the quantitative screening of pollen donors with different ploidy levels, based on the fertilization success of the maternal plant, and 3) for the screening of parents producing a high proportion of polyhaploids.

Genetic diversity and reproductive biology in ecotypes of the facultative apomict Hypericum perforatum L.

Apomixis is a mode of asexual reproduction through seed. Progeny produced by apomixis are clonal replicas of a mother plant. The essential feature of apomixis is that embryo sacs and embryos are produced in ovules without meiotic reduction or egg cell fertilisation. Thus, apomixis fixes successful gene combinations and propagates high fitness genotypes across generations. A more profound knowledge of the mechanisms that regulate reproductive events in plants would contribute fundamentally to understanding the evolution and genetic control of apomixis. Molecular markers were used to determine levels of genetic variation within and relationship among ecotypes of the facultative apomict Hypericum perforatum L. (2n = 4x = 32). All ecotypes were polyclonal, being not dominated by a single genotype, and characterised by different levels of differentiation among multilocus genotypes. Flow cytometric analysis of seeds indicated that all ecotypes were facultatively apomictic, with varying degrees of apomixis and sexuality. Seeds set by haploid parthenogenesis and/or by fertilisation of aposporic egg cells were detected in most populations. The occurrence of both dihaploids and hexaploids indicates that apospory and parthenogenesis may be developmentally uncoupled and supports two distinct genetic factors controlling apospory and parthenogenesis in this species. Cyto-embryological analysis showed that meiotic and aposporic processes do initiate within the same ovule: the aposporic initial often appeared evident at the time of megaspore mother cell differentiation. Our observations suggest that the egg cell exists in an active metabolic state before pollination, and that its parthenogenetic activation leading to embryo formation may occur before fertilisation and endosperm initiation.

Developmental expression of ASG-1 during gametogenesis in apomictic guinea grass ( Panicum maximum)

We have used Western blue-visualized in situ-hybridization (ISH) to monitor the expression of apomixis-specific gene-1 (ASG-1, GenBank accession number AB000809) during gametogenesis in obligate-sexual and facultative-apomictic (aposporic) genotypes of guinea grass (Panicum maximum). The in situ-analysis revealed that ASG-1 is not expressed in the ovule during early floral development in both, the facultative apomicts (A1 stage) and the obligate sexuals (S1 stage). With the appearance of the aposporous initial cell(s) in the ovule of the apomictic type (A2-1 stage), ASG-1 expression is strong and specific to this apomixis-specific cell. ASG-1 expression continued through different stages of aposporous embryo sac development (A2-2 stage), indicating that the gene may play a role in this developmental process. Regular embryo sacs in sexual types did not show hybridization signals (S2 stage). However, strong ASG-1 expression was detected in immature pollen grains and young embryos in both reproductive types, suggesting that ASG-1 may be an allele derived from the obligate-sexual wild type. Expression in pollen grains faded with maturation. In a heterologous system, using Paspalum notatum, a facultative-aposporic tropical grass (bahia grass), identical results were obtained. The results are discussed in view of the fact, that ASG-1 shows some homology to genes known to be seed- or embryo-specific or involved in processes related to cell growth.

Preliminary Isolation of Viable Protoplasts from Egg Cells in Facultative Apomictic Guineagrass (Panicum maximum)

Preliminary Isolation of Viable Protoplasts from Egg Cells in Facultative Apomictic Guineagrass (Panicum maximum)

Demonstration of linkage and development of the first low-density genetic map of garlic, based on AFLP markers

Garlic (Allium sativum L.) is a long-cultivated, clonally propagated diploid plant (2n=2x=16). With routine seed production now underway, we used populations (MP1 and MP2) generated by self-pollination of unrelated plants to generate two low-density genetic maps of garlic, consisting of amplified fragment length polymorphism (AFLP) and gene-specific markers. We did not observe any two plants with identical marker patterns in either population, indicating that they were the result of amphimixis rather than apomixis. This is an important finding, since several Alliums are facultative apomicts. A total of 360 markers segregated in MP1 (12.8 AFLP markers per primer combination) and 321 markers segregated in MP2 (13.9 per primer combination) to indicate a fairly high level of genetic heterozygosity in the garlic nuclear genome. Of these markers, 15.3% in MP1 and 24.3% in MP2 had segregation ratios distorted from the expected 3:1. Interestingly, 94.7% of those distorted segregations fit a 15:1 segregation ratio for duplicated loci, suggesting extensive levels of duplication in the garlic genome and supporting similar observations for onion. The genetic map for the MP1 family with 216 markers spanned 1,166 cM of the garlic genome (5.4 cM average), while 143 markers of MP2 spanned 862 cM (6.0 cM average). Gene-specific markers for alliinase, chitinase, sucrose 1-fructosyltransferase (SST-1), and chalcone synthase (CHS) were mapped, demonstrating the immediate utility of the garlic genetic map. These two garlic families had relatively few segregating AFLP markers in common, which supports their relatively distant relationship based on diversity analysis. Of those markers that were conserved, linkages were also conserved.

Breeding systems and continuing evolution in the endemic Sorbus taxa on Arran.

The Arran whitebeams Sorbus arranensis and S. pseudofennica are two endemic woody plant taxa that have evolved on Arran through hybridisation. S. arranensis is a triploid hybrid between the widespread diploid S. aucuparia and the rare tetraploid S. rupicola. S. pseudofennica is a tetraploid formed by crossing between S. arranensis and S. aucuparia. In order to determine the mating systems of the two endemic species six maternal trees of each taxon together with 10-12 of their seed offspring were scored for their phenotype at three microsatellite loci and one nuclear intron locus. All seeds of S. arranensis were identical in phenotype to their maternal parents. In S. pseudofennica, 17.5% of all seeds differed in marker phenotype from their maternal parent. The proportion of seed with nonmaternal phenotypes varied significantly among maternal trees of S. pseudofennica. The results suggest that the triploid S. arranensis is an obligate apomict, whereas the tetraploid S. pseudofennica is a facultative apomict. Molecular marker analysis of three trees from Arran with an unusual leaf morphology indicates that they are the product of sexual reproduction by S. pseudofennica, and may originate from hybridisation with S. aucuparia. This research demonstrates that the Sorbus taxa on Arran are participants in an active evolutionary process generating novel biodiversity. Conservation programmes for these taxa should aim to preserve this evolutionary process rather than the individual taxonomic entities that it produces.

The role of tetraploids in the sexual-asexual cycle in dandelions (Taraxacum).

Apomictic plants often produce pollen that can function in crosses with related sexuals. Moreover, facultative apomicts can produce some sexual offspring. In dandelions, Taraxacum, a sexual-asexual cycle between diploid sexuals and triploid apomicts, has been described, based on experimental crosses and population genetic studies. Little is known about the actual hybridization processes in nature. We therefore studied the sexual-asexual cycle in a mixed dandelion population in the Netherlands. In this population, the frequencies of sexual diploids and triploids were 0.31 and 0.68, respectively. In addition, less than 1% tetraploids were detected. Diploids were strict sexuals, triploids were obligate apomicts, but tetraploids were most often only partly apomictic, lacking certain elements of apomixis. Tetraploid seed fertility in the field was significantly lower than that of apomictic triploids. Field-pollinated sexual diploids produced on average less than 2% polyploid offspring, implying that the effect of hybridization in the 2x-3x cycle in Taraxacum will be low. Until now, 2x-3x crosses were assumed to be the main pathway of new formation of triploid apomicts in the sexual-asexual cycle in Taraxacum. However, tetraploid pollen donors produced 28 times more triploid offspring in experimental crosses with diploid sexuals than triploid pollen donors. Rare tetraploids may therefore act as an important bridge in the formation of new triploid apomicts.

Sexuality and no Apomixis Found in Crossing Experiments with Diploid Potentilla Argentea

Thirty-six crosses between plants of the putative facultative apomictic diploid Pofentilkz argentea L. (s.l.) from southern Sweden led to the production of hybrid offspring, as determined by isozyme analysis. Forty-three crosses with diploid P. argentea as mother plants and hexaploid P. argenrea L. (s. l.) or hexaploid P. collina Wib. as pollen donors resulted in normal-looking seeds but with no, or only low, rates of germination. Among the few offspring obtained, two were found to be tetraploid as determined by Row cytometry. Offspring plants with non-hybrid genotypes were occasionally obtained in the crosses, which in all cases could be explained as being due to selfing resulting from incomplete emasculations or unwanted insect pollinations. We conclude that, on the basis of currently available information, diploid Potentilla argentea is not a facultative apomict.